Scaling up enzyme purification and recovery methods for large scale production is often very difficult. Many of the processes which are practical in small scale enzyme preparations are impractical, uneconomical, and too labor-intensive for large scale production.
For example, it is known that alcohol oxidase can be recovered from Pichia pastoris grown on methanol. In small scale preparations, alcohol oxidase is recovered by homogenizing Pichia pastoris cells grown at high cell density (85-150 grams of biomass dry weight per liter) by bead milling followed by filtration or centrifugation to remove cell solids, yielding a crude solution containing alcohol oxidase. The crude solution is then dialyzed or ultrafiltered with a final ionic concentration of between 0.05M and 0.01M and a pH of from about 5.75 to about 6.75 to yield crystalline alcohol oxidase. However, this process is only suitable for small scale preparation with a beginning volume of about 10 liters.
To scale up this small scale alcohol oxidase preparation, a number of difficulties must be overcome. First, Pichia pastoris is preferably grown at high cell densities between 85-150 grams of biomass dry weight per liter. This cell density may not be suitable for some processing steps, but it is desirable to maintain because of the higher initial concentration of alcohol oxidase it may provide. This higher initial concentration of alcohol oxidase, if maintained, may result in higher yields and lower volumes of material to process. Second, since alcohol oxidase is a large enzyme, being approximately 650,000 daltons in size, there is very little literature guidance as to what operating parameters may be appropriate for an enzyme of this size. Further, the literature appears to indicate that filtration, such as crossflow filtration, may provide low yields of an enzyme of alcohol oxidase's size. Finally, scaling up the preparation of alcohol oxidase must be controlled so that acceptable purity, yield, and specific activity of the alcohol oxidase can be obtained.
Thus, it would be a significant contribution to the art to develop a large scale process for the purification of alcohol oxidase in high purity.
Further, it would be advantageous if the large scale process for the purification of alcohol oxidase utilized conventional large scale filtration devices while providing high alcohol oxidase yields.
Additionally, it would be advantageous if the specific activity of the alcohol oxidase purified on large scale remained at levels as high as those obtained from small scale purification preparations.
Thus, it is an object of the present invention to provide a large scale process for the purification of alcohol oxidase of high purity.
It is a further object of the present invention to provide a large scale process for the purification of alcohol oxidase in high yield utilizing conventional large scale filtration devices.
It is another object of the present invention to provide a process to purify alcohol oxidase while maintaining high levels of alcohol oxidase specific activity.
Other aspects, objects, and several advantages of this invention will be apparent from the specification, examples, and claims.